This Program Project applies an extraordinarily broad array of approaches to understand the integrated behavior across time scales and from atomic resolution to whole animals of the nuclear factor kappa B (NF?B) family of transcription factor signaling system. NF?Bs control cellular stress responses, cell growth, survival, and apoptosis. System control is accomplished by interaction a family of inhibitors of kappa B proteins (I?Bs) that sequester NF?B family members in the cytoplasm poised for rapid activation. Experiments and mathematical modeling showed that rapid degradation of free inhibitors achieves low free inhibitor concentrations and robust signal response. Coupled folding and binding of regions of the proteins appears critical for defining degradation rates and binding kinetics. In Overall AIM 1, we will explore how the degradation rate of the canonical inhibitors controls signaling. Folding kinetics by stopped flow and T-jump, theoretical studies on the folding pathways, NMR dynamics, and identification of the degrons will together address this aim. In Overall AIM 2, we will explore ways in which the signaling is under kinetic control. We have discovered that I?B? facilitates dissociation of NF?B from transcription sites (stripping). This phenomenon will be analyzed in cells using mutants deficient in stripping, the mechanism will be predicted by theoretical studies, the kinetics will be measured by single molecule studies, the structures of ternary complexes will be studied by NMR and the effects of stochasticity on the kinetics of transcription activation will be incorporated. In Overall AIM 3, we will explore the idea that I?Bs stabilize certain NF?B homo and heterodimers affecting the specificity of stimulus response. Certain complexes activate specific genes, yet the molecular mechanism, binding affinities, foldedness of the inhibitors, and roles in cells are still incomplete. Our multiscale, quantitative combination of theory, in vitro biochemical and biophysical characterization, and in vivo studies will enable us to map the landscape by systematic perturbation of the protein interaction dynamics can be quantitatively linked to the emergent biological response.